Affinity chromatography (Protein A/G)

Affinity – IEX: 

Herrmann (WO 2005/100394A2) discloses a process for purifying IL-15/Fc fusion protein by applying the composition to an affinity chromatography column and applying the eluate to an ion exchange chromatography column. Additionally, the eluate from the ion exchange can be applied to a gel filtration column or to a hydrophobic interaction chromatography column.

Herrman (WO 2005/100394) discloses a process for purifying a fusion protein by applying an affinity chromatography, applying the eluate to an ion exchange chromatography.

Protein A -AEX:

Treyo (US 14/775,992, published as US 2016/0024144; see aslo US 16/042,965, published as US 2019/0085021) discloses a method for purifying a recombinant prtoein such as atumor necrosis factor receptor Fc fusion protein.  such as etanercept. 

–Protein A- AEX –HIC

Kulkarni (US2014/0128577) discloses a method for purificaiton of TNFR:Fc fusion protein compirising Protein A chromatography, HIC in bind and elute mode and AEX which is performed in bind elute mode. In one embodiment AEX is performed prior to the HIC.  

Majumder (WO 2014/102814) disclsoes purification of a TNFR-Fc fusion protein which includes the scheme of Protein A chromatography, AEX  (Q Sepharose FF), HIC (the elute of Q Sepharose FF is added to 0.8 M of ammonium sulphage and the sample after filtraiton is laoded on phenyl sepharose resin preequilibrated with 20 mM Phosphate buffer containing 0.8 M ammonium sulphage. After washing with the equilibraiton buffer the protein is eluted wither with 20 mM Phosphate buffer contnaining 0.3 M ammonium sulphage buffer in step gradient mode or with 20 mM Phosphate buffer in linear gradien mode) HIC output is then diafiltrated and concentration against a suitable buffer followed by nano filtration. . 

–—-Protein A -AEX -HIC -CEX

karur (US 16/083891, published as US 2020/0283472; see also wO 2017/168296) disclsoes a method of purifying an Fc fusion protein such as etanercept (TNF fusion with Fc of IgG1) which includes the steps of AEX in bind and elute mode, HIC using polypropylene glycol (PPG) chromatography in bind and elute mode, followed by CEX in bind and elute mode. 

Protein A – CEX: 

Eon-Duval (US 12/377122) discloses a process for reducing the content of free Fc moeties in a fluid comprising an Fc containing protein (exemplifies a TACI-Fc fusion protein) comprising an affinity A step followed by a strong cation step on Fractogel EMD SO3- which includes a wash step with a first buffer having a conductivity of 8.2 to 9.2 mS/cm and a pH of 6.0 to 7.0 to remove free Fc-moieites. 

A packed bed CEX following Protein a chromatography typicaly has yields of greater than 805 and aggregate levels of less than 0.5%. By contract, an expanded bed chromatography reduced aggregates in yields of between 50-60% and only at the lwoest condutivities with each pH was the level of aggreggates reduced to below 1%. Blank (Bioseparation 10: 65-71, 2001)

Protein A -CEX -AEX:

Nti-Gyabaah (US 2014/0187751) discloses methods for purifying a Fc fusion protein proudced in a eukaryotic system and in particular TNFR-Fc (e.g., Etanercept) which includes the steps of Protein A affinity chromaotgoraphy followed by two ion exchange chromatography steps of CEX followed by AEX both in bind-and-elute modes. 

Protein A/G – CEX – AEX – HA: 

Eon-Duval (WO2008/025747) also discloses a process for the purification of an Fc-fusion protein comprising the steps (1) Protein A/G affinity chromatography, carrying out the elution with a pH gradient, preferably from pH 4.5 to 2.8 and then subjecting the eluate to CEX, subjecting the eluate to AEX and subjecting the flow through to hydroxyapatite chromatography. 

Protein A — HA:

Vedantham (US 7,476,722) discloses purification of a protein that may comprise an Fc porition of an antibody such as TNFR:Fc and a second protein such as Protein A /G using affinity chromatography using the second protein affixed to the solid support as an adsorbent followed by hydroxyapatite in flow through mode. 

Protein A- HIC

Kulkarni (WO 2012/176158) discloses a method of purifying a TNFR:Fc fusion protein that includes the steps of Protein A followed by hydrophobic interaction chromatography in bind and elute mode where the buffer solution does not contain any additives and is at a conductivity of 25-40 mS/cm followed by AEX.

–Protein A -HIC-AEX-Mixed Mode

Banerjee (US 2017/0152298 disclsoes purificaiton of TNFR:Fc fusion prtein by Protein A in bind-elute mode followed by HIC in bind and elute mode followed by AEX in bind -elute mode and then mixed-mode chromatogrpahy in flow through mode.

Protein A -Mixed Mode -HIC:

–Multimodal Anion Exchange:

Keller (WO 2016/009049) discloses a method for purifying a TNFR-Fc fusion protein which includes the steps of subjecting a solution that includes TNFR-Fc to affinity chromatography, optionally subjecting the eluate to AEX, subjecting the eluate to multimodal anion exchange chromatography and then subjecting the flow-through to HIC and collecting the eluate to botain purified TNFR-Fc. 

Hydrophobic Interaction Chromatography (HIC):

Ghil (US 2018/0079796) disclsoes a method of prearing human 75 kDa (p75) TNFR fused to an Fc region of human IgG1 using HIC such that the anti-TNF-alpha polypetpide binds to the HIC regin and eluting the anti-TNF alpha polypetpide. Etanercep is the recombinant human tumor necrossis factor receptor p75Fc fusion protein. Etanercept is well establisehd and widely used in clinical practice. 

Park (US 2017/0369553) discloses a method for preparing a TNFR-Fc fusion proteins which includes the steps of laoding a sample containing a TNFR-Fc fusion protein mixture produced form mammalian cells into a column filled with HIC comprsing an aromatic functional group, which is pre-equilibration with an equilibraiton buffer comprising sodium chloride or ammonium sulfate and collecting an eluate with an elution buffer comprising soidum chloride or ammonium sulfate at the same concentraiton as that of the equilibraiton buffer. 

Won (US 2014/0316114) discloses a method for preparting a TNFR-Fc fusion proteins which includes loading a sample which includes the TNFR-Fc produced in mammalian cells to the HIC column pre-equilibrated with an equilibration buffer that includes one or mroe salts selected form sodium citrate, sodium sulfate and sodium phosphate in an amount of 10-14 gL bed per volume of chromatography resin, washing the column with the same salt as the equilibraiton buffer to remove clipped forms of TNFR-Fc fusion protein and eluting the active TNFr-Fc fuison proteins from the column with an elution buffer have salt concentraiton lower than the equilibration buffer. 

HIC-Pre-filter-Virus filter:

Sharma (US Patent Application 16/708,987, published as US 202010190137) discloses purification of the TNFR-Fc fusion prtoein etanercept which includes HIC (e.g., Toyopearl Butyl-650 M column) and subjecting the elution pool to virus filtration (e.g., VireSolve Pro viral filter). In one embodiment, a prefileter (Viresolve Prefilter A1HC) placed upstream of the viral filtration membrane reduced flux decay.

Hydroxyapatite

For purifying Antibody drug conjugate (ADCs);

Lin (US 216/0051695) discloses purification of an anti-HER2-IgG1 conjugated to maleiminocapyroyl drug using HA with a wash and elution buffer that includes sodium phosphate buffer. 

Nadkarni (US 15/778, 290 published as 15/778,290) discloses purifcation of an ADC in monomer form using HA that employs a low sodium phosphate buffer wash and a high or tradient increasing sodium phosphate buffer. 

Ion Exchange

Cation-Exchange (CEX): CEX removed antibody aggregates. Antibody aggregates are more tightly bound than the antibody monomer and elute after the main peak. Elution conditions must be optimized to elute all the antibody monomer but not the aggregated antibody. Blank (Bioseparation 10: 65-71, 2001)

Mixed Mode Chromatography: 

Mazzola (US 2005/0031627) discloses purification of an antibody-DM1 conjugate with ceramic hydroyapatite column as well as ion exchange chromatography. Dai (US2007/0048314A1) discloses purification of an antibody-agent conjugate to an mixed mode ion exchange chromatography. Snyder US 12/769460) discloses a method of purifying an antibody-agent conjugate wherein the agent comprises the label phycoerythrin using a contacting step comprising an increased concentration of salt and elutuing step comprising increasing the concentration of the buffer. 

TNFR-Fc Fusion:

Banerjee (US 2017/0152298) discloses TNFR-Fc fusion prtoein purificaiton using mixed-mode chromatogrdaphy in flow thorugh mode. In another embodiment, Banerjee disclsoes purifying TNFR-Fc fusion protein which includes the steps of affinity chromatography, mixed mode chromatography in bind and elute mode. In a separate emboidment, the process for purifying TNFR-Fc fusion protein includes the steps of affinity chromatography, HIC in bind an delute mode and then mixed mode chromatogrpahy in bind and elute mode. 

Filtration (See also filtration for antibody purification)

Yamaguchi (US2010/0190965) discloses a method of separating immunoglobulin monomers from aggregates using cross-flow filtration with an ultafiltration membrane. The immungolobulin concnetration is 1-150 g/L and cross flow filtration is performed using an UF membrane having a MWCO of 100k or more and less than 500k so that immunoglobulin monomers pass through the UF membrane with a permeability of 80% or more while achieving a factionation performance in which the permeability ratio of immunoglobulin dimers to monomers that pass throguh the UF membrane is 0.20 or less. 

Tangential flow filtration: Dai (WO/2007/024536) discloses a process for preparing a conjugate comprising a cell binding agent chemically coupled to a drug by contacting the cell binding agent with a bifunctional crosslinking reagent to covalently attach a linker to the cell binding agent, subjecting the first mixture to TFF to prepare a first mixture of cell bindin agents haivng linkers bound thereto and conjugating a drug to the cell binding agents having linkers by reacting them with a drug  in a solution having a pH of about 4-9 to preapre a second mixture comprising a cell binding agent chemically coupled to the linker to the drug, free drug and reaction by products and subjecting hte second mixture to TF to purify the cell binding agents chemically coupled through the linkers to the drug.